Abstract Type IX collagen is a quantitatively minor component of cartilage collagen fibrils. Although a few mutations have been associated with multiple epiphyseal dysplasia, recent evidence suggests involvement of type IX collagen in a wider spectrum of phenotypes. The functional role of this molecule remains undetermined, in part due to difficulties in obtaining high amounts of intact protein. To facilitate more efficient mutation screening and comparison of the genomic organization of the human genes encoding the α1(IX), α2(IX) and α3(IX) polypeptides, their genomic structures were characterized. Complete nucleotide sequences were determined for the COL9A2 and COL9A3 genes along with sequences for all the exon boundaries in the COL9A1 gene. Putative transcription control elements were identified and the alternative promoter region was characterized in the human and mouse COL9A1 genes. Mutation screening was performed for the COL9A3 gene and two apparently neutral 9-bp deletions within the COL1 domain were identified. These are the first deletions within a triple-helical domain of any collagen that are not associated with a disease phenotype. An insect cell expression system with an exogenous source of prolyl 4-hydroxylase was used to produce heterotrimeric human type IX collagen. The recombinant protein consisted of the three a chains in a 1:1:1 ratio and showed correct folding and high thermal stability. Up to 10 mg of secreted protein could be purified from a litre of culture medium. The expression system was used to analyze the chain association of type IX collagen in cellulo. Although the chains are capable of homotrimerization, a preference for heterotrimer formation was noted.The neutral deletion was characterized further using the insect cell system. Mutant α3(IX) chains carrying a deletion of one Gly-X-Y triplet were shown to form correctly folded heterotrimers with the wild-type α1(IX) and α2(IX) chains. The results suggest a function for the NC2 domain in neutralizing the effect of the deletion. This work provides a novel means for the analysis of type IX collagen mutations and their protein-level effects, and should enable future studies to be made of the structure-function relationship in type IX collagen.

Abstract Collagens IX and XI are quantitatively minor components of the collagen fibrils in cartilage. The spectrum of the phenotypes caused by mutations in the COL2A1 gene coding for collagen II, the main cartilage collagen, is relatively well defined, but there is little data on the phenotypes caused by collagen IX and XI mutations. The structure of the human COL11A1 gene coding for the 1 chain of collagen XI was characterized here. It was found to consist of 68 exons and span 160 kb, excluding introns 1 and 4. Over 50 kb of new intronic sequences were defined. The exon-intron organization coding for the major triple helical domain was found to be identical to that of the human COL11A2 gene, which codes for the 2 chain of collagen XI. The sensitivity of conformation sensitive gel electrophoresis (CSGE) for mutation detection was improved and tested with a large number of sequence variations in collagen genes. The sensitivity with the revised conditions was found to be close to 100%. In addition, CSGE was found to be a simple and practical method for analyzing large numbers of samples. Fifteen mutations in the COL11A1 gene and eight in the COL2A1 gene were found by CSGE in patients with Marshall or Stickler syndrome. The genotypic-phenotypic comparison indicated that mutations leading to a premature translation termination codon in the COL2A1 gene resulted in Stickler syndrome and splicing mutations of 54 bp exons in the C terminal half of the COL11A1 gene resulted in Marshall syndrome. The other COL11A1 mutations caused phenotypes overlapping both syndromes. In an analysis of the COL9A2 gene in 157 patients with intervertebral disc disease, six were found to have a tryptophan for glutamine substitution in the central collagenous domain of the collagen IX molecule. None of 174 control individuals had this substitution. The substitution cosegregated with the phenotype in the families studied, and linkage and linkage disequilibrium analyses supported the association of the locus and the disease with a joint lod score of over 11.

M.Sc. (Med.), Faculty of Health Sciences, University of the Witwatersrand, 2009. / Haemophilia A is a common X-linked recessive bleeding disorder, affecting about 1 in 5000 males worldwide. It is caused by a deficiency of functional coagulation Factor VIII (FVIII), resulting in prolonged or abnormal bleeding episodes. The severity of the disease is related to the level of functional FVIII in the plasma. The FVIII gene is a large gene, located at Xq28 with a complex genomic organisation. It contains 26 exons spanning 186kb of genomic DNA, and produces a 9kb transcript, resulting in a functional protein of 2332 amino acids. Over 900 mutations, which span a wide variety of categories, including rearrangements; complete or partial gene deletions; large insertions; duplications; frameshift mutations; splicing defects; nonsense and missense mutations, have been identified in the FVIII gene. Most mutations are rare or family specific, except for the intron 22 inversion mutation, which is reported to account for 45-50% of mutations in severe haemophilia A patients in most populations. A second inversion mutation, in intron 1, accounts for approximately 3.8% of haemophilia A patients in the UK. In South Africa, diagnostic mutation testing is currently only available for the intron 22 inversion mutation. Linked marker analysis is used to track high risk alleles in families where the disease-causing mutation is unknown. This study aims to evaluate an mRNA-based method to identify disease-causing mutations in South African haemophilia A patients and improve the diagnostic service. Blood samples from 120 patients were tested first for the intron 22 and then for intron 1 inversion mutations. Inversion negative patients were analysed further using mRNA. A mutation has been identified in 73.3% (88/120) of all patients. 30% (36/120) of patients had the intron 22 inversion, 2.5% (3/120) an intron 1 inversion and 40.8% (49/120) of patients had a mutation identified by mRNA analysis. A mutation was not identified in the remaining 26.7% (32/120) due to sample and technical difficulties. Of the 49 mutations identified through mRNA analysis, 28 patients (57.1%) have a point mutation (17 missense (34.7%), 9 nonsense (18.4%) and 2 splice-site mutations (4.1%)), 9 patients (18.4%) have a deletion and 7 patients (14.3%) have an insertion. Another 5 patients (10.2%) have a complex mutation (including patients where an exon deletion was detected on mRNA analysis, but no mutation was identified on DNA analysis). One mutation, c.3637insA, was found recurrently in 14% (6/43) of patients from the white population. This single base insertion results in a frameshift mutation with a premature stop codon at amino acid 1221 (only translating about half the normal FVIII protein). This common mutation, together with haplotype analysis, suggests a founder effect for this mutation. mRNA analysis of the FVIII gene is a novel technique in mutation detection for haemophilia A. It decreases the costs involved in sequencing the coding region and it offers improved mutation detection compared to DNA analysis. Diagnostic testing in South Africa should be extended from the current intron 22 inversion mutation to include DNA analysis for the intron 1 inversion and the founder mutation (c.3637insA) in white patients, followed by mRNA testing, starting with the analysis of the fragments spanning exon 14. mRNA analysis identifies an additional 55.7% of mutations compared to conventional diagnostic testing for the intron 22 inversion alone.

Inherited retinal and corneal dystrophies represent a group of disorders with great genetic heterogeneity. Over 250 genes are associated with retinal diseases and 16 genes are causative of corneal dystrophies. This thesis is focused on finding the genetic causes of corneal dystrophy, Leber congenital amaurosis (LCA), Stargardt disease and retinitis pigmentosa in families from northern Sweden.  By whole exome sequencing a novel mutation, c.2816C>T, p.Thr939Ile, in Collagen Type XVII, Alpha 1 chain, COL17A1, gene was identified in several families with epithelial recurrent erosion dystrophy (ERED). We showed that the COL17A1 protein is expressed in the basement membrane of the cornea, explaining the mutation involvement in the corneal symptoms. We could link all the families in this study to a couple born in the late 1700s confirming a founder mutation in northern Sweden. Our finding highlights role of COL17A1 in ERED and suggests screening of this gene in patients with similar phenotype worldwide. Furthermore the genetic causes in several retinal degenerations were identified. In one family with two recessive disorders, LCA and Stargardt disease, a novel stop mutation, c.2557C>T, p.Gln853Stop, was detected in all LCA patients. In the Stargardt patients two intronic variants, the novel c.4773+3A>G and c.5461-10T>C, were detected in the ABCA4 gene. One individual was homozygous for the known variant c.5461-10T>C and the other one was compound heterozygote with both variants present. Both variants, c.4773+3A>G and c.5461-10T>C caused exon skipping in HEK293T cells demonstrated by in vitro splice assay, proving their pathogenicity in Stargardt disease. Finally, in recessive retinitis pigmentosa, Bothnia Dystrophy (BD), we identified a second mutation in the RLBP1 gene, c.677T>A, p.Met226Lys. Thus, BD is caused not only by common c.700C>T variant but also by homozygosity of c.677T>A or compound heterozygosity. Notably, known variant, c.40C>T, p.R14W in the CAIV gene associated with a dominant retinal dystrophy RP17 was detected in one of the compound BD heterozygote and his unaffected mother. This variant appears to be a benign variant in the population of northern Sweden. In conclusion, novel genetic causes of retinal dystrophies in northern Sweden were found demonstrating the heterogeneity and complexity of retinal diseases. Identification of the genetic defect in COL17A1 in the corneal dystrophy contributes to understanding ERED pathogenesis and encourages refinement of IC3D classification. Our results provide valuable information for future molecular testing and genetic counselling of the families.

Treatment success often depends on the availability of accurate and reliable diagnostic assays to guide clinical practitioners in their treatment choices. An optimal test must excel in specificity and sensitivity, and depending on the application area time, low-cost and simplicity are equally important. For instance, time is essential in infectious diagnostics but this is less important in non-invasive prenatal testing (NIPT). In NIPT, specificity and sensitivity are the most important parameters. In this thesis I describe the development of four different methods, all based on padlock probes and rolling circle amplification, intended for molecular diagnostics. Application areas range from infectious disease diagnostics to NIPT and oncology. The methods described have in common that they overcome certain limitations of currently available assays. This thesis includes two new assays targeting infectious agents: one assay specifically detecting a highly variable double stranded RNA virus and the second assay demonstrating a new format of antibiotic susceptibility testing, which is rapid and generally applicable to different pathogens. Furthermore, I describe the development of a method that uses methylation markers to enrich fetal DNA, accurately quantify chromosome ratios and thus, detecting trisomy 21 and 18. The fourth method described in this thesis uses gap-fill ligation of padlock probes to detect diagnostic relevant point mutations with high specificity in situ. The assays presented have the potential, after automation and successful validation and verification studies, to be implemented into clinical practice. Furthermore, these assays demonstrate the wide applicability of padlock probes which, due to their properties in regard to specificity and multiplexity, are useful tools for nucleic acid detection in vitro as well as in situ. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

This thesis demonstrates the application of biosensor technology for molecular biology investigations, utilizing a surface plasmon resonance based optical device for mass sensitive detection of biomolecular interactions at a chipsurface. Oligonucleotide model systems were designed for analysis of the action of DNA manipulating enzymes. DNA ligation, DNA cleavage and DNA synthesis could be quantitatively monitored in real-time. A protocol for DNA minisequencing was also established based on prevention of chain elongation by incorporation of chain-terminators. Determinations of affinities for short oligonucleotides hybridizing to an immobilized target were performed with various sequence content, length, temperature and degree of complementarity. The decrease in affinity for hybridizations involving mismatch situations was found to be strongly dependent on the relative position of the mismatch. Interestingly, also end-mismatches were clearly detectable. The stabilization effect achieved upon co-hybridization of two adjacently annealing short oligonucleotide modules (modular primer effect) was also investigated for different module combinations and hybridization situations. The modular concept of hybridizations was subsequently demonstrated to result in enhanced Capture of single stranded PCR products. The sequence based DNA analysis, first introduced with oligonucleotide modelsystems, was extended to the scanning and screening formutations in PCR amplified DNA from clinically relevant samples. Several different formats were investigated, eitherwith the PCR products immobilized on the chip and oligonucleotides injected or vice versa. Again, mismatch discrimination could be observed for wild type and mutant specific oligonucleotides hybridizing to the targets. The experimental set-up for mutation detection was further developed by the introduction of a subtractive mismatch sensitive hybridization outside the instrument and a subsequent determination of the relative amounts of remain ingoligonucleotides with analytical biosensor monitoring of hybridizations between fully complementary oligonucleotides. In conclusion, the applied technology was found to be a suitable tool for a wide range of molecular biology applications, with emphasis on hybridization analysis and mutation detection. / QC 20100611

This thesis reports design, fabrication, and implementation of two different micro electrophoresis system architectures for DNA analyses. The first architecture is traditional single channel layout with several design alternatives for size-based separation of DNA fragments. The second one is novel double channel architecture specialized for rapid mutation detection using heteroduplex analysis (HDA) method with an application of a newly designed injection technique. Besides achieving high resolution separations within the length of 1 mm with single channel arrangement, HDA was successfully applied for 590 base pair (bp) long PCR sample with 3 bp mutations in a separation length of 50 &micro / m in less than 3 minutes with double channel structure. Microchannels were formed using parylene-C due to its conformal deposition, no surface treatment requirement, transparency, biocompatibility, low background fluorescence, etc. Using the advantage of parylene in fabrication, the microchannels were fabricated with an only three-mask process. New double channel architecture is obtained by dividing the 200 &micro / m-wide separation channel into two parts by a 20 &micro / m-thick wall between them. For sample injection, various techniques, such as traditional cross, double-T, and double-L were investigated and optimized for single channel architecture assisting with pullback injection method. For double channel architecture, a novel, u-turn injection technique was applied. Precise control of sample amount by adjusting the injection time was accomplished by this new technique. Using high resolution cross-linked polyacrylamide gel as sieving material, separations were achieved in a very short length and time. Electrophoresis was performed in both channels of the double channel microchips simultaneously under the same conditions. This gives the chance of having a control channel in microchip format, which is very critical for the accuracy and reliability of the results in genetic analyses.

Leber’s hereditary optic neuropathy (LHON) is the most commonly inherited disease that causes blindness in one or both eyes, with a minimum prevalence of 1 in 31 000 in the northeast of England. What causes LHON is not fully known but three mitochondrial mutations, G3460A, G11778A, and T14484C, have been identified in over 95 % of all LHON patients. To diagnose LHON, detection methods like sequencing, allele specific polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) are used to identify these three mutations. The methods are now evolving into multiplex ones to increase efficiency, the aim of this study was therefore to optimize one of them, a multiplex PCR-RFLP method developed in 2015. This study was however not completed, due to the COVID-19 pandemic, but a series of preparatory steps were performed before its termination. DNA extraction was performed, both genomic and plasmid, using a kit and in-house protocols. The DNA was then used for polymerase chain reactions (PCRs), for both the human β-globin gene and for the three mutations, where magnesium concentration and annealing temperature was optimized. This study resulted in clear, high quality extractions, with the kit as the preferable method. It also indicated that a 3 mM magnesium concentration and an annealing temperature of 59 °C was optimal for all mutations when using so called LHON primers. The conditions for the PCR using the multiplex primers might be different, therefore a new study is required to evaluate the multiplex PCR-RFLP method further. / Bakgrund: Lebers hereditära optikusneuropati (LHON) är en vanlig ärftlig sjukdom som orsakar blindhet. LHON orsakas i över 95 % av fallen av en av tre mitokondriella mutationer, där en byggsten i mitokondriens DNA felaktigt bytts ut mot en annan. Dessa mutationer heter G3460A, G11778A och T14484C. För att diagnostisera sjukdomen detekteras mutationerna, bland annat genom att extrahera DNA från blod, DNA som man sedan skapar otaliga kopior av genom en metod som heter ”polymerase chain reaction” (PCR). Dessa kopior kan sedan klyvas i bitar med hjälp av enzym och baserat på fragmentens storlek kan det avgöras om personen har mutationen eller inte, detta kallas för ”restriction fragment length polymorphism” (RFLP). I nuläget letar man efter en mutation i taget men det har utvecklats några metoder där man kan hitta alla mutationer på en gång och den här studiens syfte var att undersöka hur man på bästa sätt kan utföra en av dessa metoder, en så kallad multiplex PCR-RFLP. Metod: Studien avbröts i förtid på grund av ett pandemiskt utbrott av COVID-19 men hann omfatta DNA-extraktion från humant blod och bakterier med hjälp av ett kommersiellt kit och laboratoriets egna protokoll. Även PCR utfördes för en normal genuppsättning och de tre mutationerna. Resultat och slutsats: Extraktionen gav bra resultat med alla metoder men det kommersiella kitet gav bäst resultat. PCR med det DNA som extraherats fungerade bara ibland vilket gjorde det svårt att dra några större slutsatser, oavsett krävs fler studier för att undersöka metoden eftersom arbetet inte kunde slutföras.

Nucleic acid technology has assumed an essential role invarious areas ofin vitrodiagnostics ranging from infectious diseasediagnosis to human genetics. An important requirement of suchmolecular methods is that they achieve high sensitivity andspecificity with a fast turnaround time in a cost-effectivemanner. To this end, in this thesis we have focused on thedevelopment of sensitive nucleic acid strategies thatfacilitate automation and high-throughput analysis. The success of nucleic acid diagnostics in the clinicalsetting depends heavily on the method used for purification ofthe nucleic acid target from biological samples. Here we havefocused on developing strategies for hybridisation capture ofsuch templates. Using biosensor technology we observed that thehybridisation efficiency could be improved using contiguousoligonucleotide probes which acted co-operatively. Byimmobilising one of the probes and annealing the second probein solution, we achieved a marked increase in target capturedue to a base stacking effect between nicked oligonucleotidesand/or due to the opening up of secondary structure. Suchco-operatively interacting modular probes were then combinedwith bio-magnetic bead technology to develop a capture systemfor the extraction of hepatitis C RNA from serum. Viral capturewith such co-operatively interacting probes extracted 2-foldmore target as capture with only a single probe achieving asimilar sensitivity to the conventional extraction protocol. Ananalogous strategy was designed to enrich for sequencingproducts prior to gel electrophoresis removing sequencingreagents and template DNA which interfere with the separationand detection of sequencing ladders, especially in the case ofcapillary gel electrophoresis. This protocol facilitates highthroughput clean-up of cycle sequencing reactions resulting inaccurate sequence data at a low cost, which is a pre-requisitefor large-scale genome sequencing products. Currently, a large effort is directed towards differentialsequencing to identify mutations or polymorphisms both in theclinical laboratory and in medical genetics. Inexpensive, highthroughput methods are therefore required to rapidly screen atarget nucleic acid for sequence based changes. In the clinicalsetting, sequence analysis of human immunodeficiency virus(HIV-1) is used to determine the presence of drug resistancemutations. Here we describe a bioluminometric pyrosequencingapproach to rapidly screen for the presence of drug resistancemutations in the protease gene of HIV-1. This sequencingstrategy can analyse the protease gene of HIV-1 from eightpatients in less than an hour and such non-gel based approachesshould be useful in the future in a clinical setting for rapid,robust mutation detection. Microarray technology facilitates large-scalemutation/polymorphism detection and here we developed amicroarray based single nucleotide polymorphism (SNP)genotyping strategy based on apyrase mediated allele specificextension (AMASE). AMASE exploits the fact that mismatchedprimers exhibit slower reaction kinetics than perfectly matchedprimers by including a nucleotide degrading enzyme (apyrase)which results in degradation of the nucleotides before themismatched primer can be extended. We have successfully typed200 genotypes (14% were incorrect without apyrase) by AMASEwhich cluster into three distinct groups representing the threepossible genotypes. In the future, AMASE on DNA microarraysshould facilitate association studies where an accuracy&gt;99%is required. <b>Keywords:</b>nucleic acid capture, modular probes,biosensor, bio-magnetic separation, hepatitis C, sequencing,pyrosequencing, mutation detection, HIV-1, drug resistance,SNP, allele-specific extension, apyrase, genotyping.

<p>Nucleic acid technology has assumed an essential role invarious areas of<i>in vitro</i>diagnostics ranging from infectious diseasediagnosis to human genetics. An important requirement of suchmolecular methods is that they achieve high sensitivity andspecificity with a fast turnaround time in a cost-effectivemanner. To this end, in this thesis we have focused on thedevelopment of sensitive nucleic acid strategies thatfacilitate automation and high-throughput analysis.</p><p>The success of nucleic acid diagnostics in the clinicalsetting depends heavily on the method used for purification ofthe nucleic acid target from biological samples. Here we havefocused on developing strategies for hybridisation capture ofsuch templates. Using biosensor technology we observed that thehybridisation efficiency could be improved using contiguousoligonucleotide probes which acted co-operatively. Byimmobilising one of the probes and annealing the second probein solution, we achieved a marked increase in target capturedue to a base stacking effect between nicked oligonucleotidesand/or due to the opening up of secondary structure. Suchco-operatively interacting modular probes were then combinedwith bio-magnetic bead technology to develop a capture systemfor the extraction of hepatitis C RNA from serum. Viral capturewith such co-operatively interacting probes extracted 2-foldmore target as capture with only a single probe achieving asimilar sensitivity to the conventional extraction protocol. Ananalogous strategy was designed to enrich for sequencingproducts prior to gel electrophoresis removing sequencingreagents and template DNA which interfere with the separationand detection of sequencing ladders, especially in the case ofcapillary gel electrophoresis. This protocol facilitates highthroughput clean-up of cycle sequencing reactions resulting inaccurate sequence data at a low cost, which is a pre-requisitefor large-scale genome sequencing products.</p><p>Currently, a large effort is directed towards differentialsequencing to identify mutations or polymorphisms both in theclinical laboratory and in medical genetics. Inexpensive, highthroughput methods are therefore required to rapidly screen atarget nucleic acid for sequence based changes. In the clinicalsetting, sequence analysis of human immunodeficiency virus(HIV-1) is used to determine the presence of drug resistancemutations. Here we describe a bioluminometric pyrosequencingapproach to rapidly screen for the presence of drug resistancemutations in the protease gene of HIV-1. This sequencingstrategy can analyse the protease gene of HIV-1 from eightpatients in less than an hour and such non-gel based approachesshould be useful in the future in a clinical setting for rapid,robust mutation detection.</p><p>Microarray technology facilitates large-scalemutation/polymorphism detection and here we developed amicroarray based single nucleotide polymorphism (SNP)genotyping strategy based on apyrase mediated allele specificextension (AMASE). AMASE exploits the fact that mismatchedprimers exhibit slower reaction kinetics than perfectly matchedprimers by including a nucleotide degrading enzyme (apyrase)which results in degradation of the nucleotides before themismatched primer can be extended. We have successfully typed200 genotypes (14% were incorrect without apyrase) by AMASEwhich cluster into three distinct groups representing the threepossible genotypes. In the future, AMASE on DNA microarraysshould facilitate association studies where an accuracy>99%is required.</p><p><b>Keywords:</b>nucleic acid capture, modular probes,biosensor, bio-magnetic separation, hepatitis C, sequencing,pyrosequencing, mutation detection, HIV-1, drug resistance,SNP, allele-specific extension, apyrase, genotyping.</p>